Groundwater pollution by halogenated, and particularly chlorinated, solvents is a worldwide problem associated primarily with industrial sites where mishandling or improper disposal has brought these solvents in contact with the soil. The most common and problematic compounds are the chlorinated ethylenes (ethenes) such as tetra- tri- or dichloroethylene. Carbon tetrachloride, chloroform and methylene chloride are also pervasive pollutants. The reasons for concern are basically threefold. First, most of these solvents are sparingly soluble in water and have the tendency to stick to soil particles. This results in tenacious underground plumes of solvent that cannot readily be removed by standard pump and treat technology (Biswas, N., et al., Water Environ. Res. 64, 170, 10, 1 (1992); Hutter, G. M., et al., Water Environ. Res. 64, 69, (1992)). Second, the toxicology of many chlorinated solvents suggests that these compounds may be carcinogenic and damaging to specific organs such as the liver and kidneys (Price, P. S., Memo of the U.S. Environmental Protection Agency, Office of Water, Washington, D.C. (1985); Vogel, T. M., Environ. Sci. Technol., 21, 722, (1987)). Finally, under conditions found in many aquifers and subsurface environments, chlorinated ethylenes and methanes are very slow to be degraded biologically. The result of these factors is that chlorinated solvents are long-lived and potentially hazardous groundwater pollutants.
Currently, there are two approaches to in situ removal of organohalogen pollutants. The first approach is the standard “pump and treat” method where groundwater is pumped to the surface for physical stripping of the contaminant from the water. For chlorinated solvents this is more of a containment method than a remediation technology, although given sufficient time (typically decades to centuries) this method may capture most of the pollutant. The other approach is biological in nature and utilizes microorganisms for the enzymatic transformation of the halogenated organics. The biological approach may utilize microorganisms indigenous to a particular site, such that the remediation process consists primarily of making additions to the contaminated site that enhance the growth of the desired microorganism. Alternatively, nonindigenous microorganisms may be introduced to a contaminated site with the necessary amendments needed for growth.
A number of organisms are known to dechlorinate persistent chlorinated pollutants. For example, Dehalobacter restrictus, Dehalospirillum multivorans, Desulfitobacterium dehalogens, and Desulfuromonas chloroethenica have been shown to partially dechlorinate chlorinated ethenes (Kochian et al., Plant Mol. Biol. 46:237 (1995); Delhaize et al., Plant Physiol. 107:315 (1995); Gerritse et al. Arch. Microbiol. 165:132 (1996); Damborsky, Folia Microbiol. (Praha) 44:247 (1999)). Similarly, Dehalococcoides ethenogenes has been shown to effect the complete dechlorination of tetrachloroethene and trichloroethene to ethene (Freedman et al., Appl. Environ. Microbiol. 55:2144 (1989)) and Maymó-Gatell et al. (Science, 176:1568 (1997)) have isolated a D. ethenogenes organism that is capable of respiratory reductive dechlorination of tetrachloroethene directly to ethene with hydrogen as an electron donor. Analysis of the 16S rRNA of the Maymó-Gatell organism revealed a unique profile that may be used to identify organisms of similar reductive capabilities.
The first step in utilizing the dechlorinating properties of the above identified organism is rapid and accurate identification. One method of identification involves the use of DNA probes (see for example in WO 89/06704, U.S. Pat. Nos. 4,851,330, and 5,574,145). Many such probes can be derived, based on the observation (see Woese, Scientific American 244(6): 98-122 (1981) for review) that parts of the 16S and 23s ribosomal RNA (rRNA) sequences vary in different species. This information was used initially for phylogenetic analyses but it has more recently been used for DNA probe-based methods for the identification of organisms. The utility of such a method is based on the conservation of nucleic acid sequence within the rRNA sequences.
Each of the cells of all life forms, except viruses, contains ribosomes and therefore ribosomal RNA. A ribosome contains three separate single strand RNA molecules, namely, a large molecule, a medium sized molecule, and a small molecule. The two larger rRNA molecules vary in size in different organisms. Ribosomal RNA is a direct gene product and is coded for by the rRNA gene. This DNA sequence is used as a template to synthesize rRNA molecules. A separate gene exists for each of the ribosomal RNA subunits. Multiple rRNA genes exist in most organisms, many higher organisms containing both nuclear and mitochondrial rRNA genes. Numerous ribosomes are present in all cells of all life forms. About 85-90 percent of the total RNA in a typical cell is rRNA. A bacterium such as E. coli contains about 104 ribosomes per cell. Much of the sequences in rRNA are highly conserved across broad evolutionary boundaries, however, certain regions are highly variable and may be used to make fine distinctions between species, sub-species and strains (U.S. Pat. No. 5,567,587).
The problem to be overcome therefore is to identify a unique 16S rDNA sequence in a bacteria capable of dechlorination of persistent chlorinated compounds for the identification and ultimate enhancement of that bacteria to remediate a contaminated site. Applicants have solved the stated problem by providing a set of nucleic acid sequences that are unique to various species of Dehalococcoides ethenogenes and other species of dehalogenating bacteria.